Circulating tumour DNA to direct the management of patients with gastrointestinal malignancies

Background Management of patients with gastrointestinal (GI) malignancies requires a personalised approach to improve survival while minimising risk. Colorectal and oesophagogastric (OG) malignancies account for 20% of all cancer deaths, with significant global socioeconomic impact. Novel technologies are required to customise treatment. Circulating tumour DNA (ctDNA) is emerging as a prognostic biomarker and predictive biomarker of treatment response and can be used to guide management. Hypotheses ctDNA can be used to predict recurrence and guide adjuvant chemotherapy (ACT) treatment decisions in patients with resected early-stage colorectal cancer (CRC) and evaluate response to therapies in metastatic CRC and OGA. Aims and objectives 1. Determine the association between detectable ctDNA and recurrence free survival (RFS) in patients with resected CRC using a tissue-free approach to ctDNA detection. 2. Assess the feasibility of using a tissue-free (versus tumour-informed) ctDNA assay to detect minimal residual disease (MRD) and guide a de-escalation strategy of ACT in patients with curatively resected CRC who are ctDNA negative post-operatively. 3. Determine the use of serial ctDNA monitoring and immune profiling to detect biomarkers of response and resistance to immunotherapy combinations in metastatic GI cancers. Methodology 1. Clinical validity of ctDNA to detect MRD based on a tissue-free ctDNA assay from TRACC Part B UK-based multi-centre observational translational study data of curatively treated CRC patients. 2. Clinical utility of ctDNA to guide adjuvant management of CRC by means of development of the TRACC Part C interventional randomised MRD study, involving additional clinical validation and process implementation and evaluation. 3. ctDNA and immune profiling in immunotherapy response monitoring in an academic phase II study of anti-PD1 and an HDAC inhibitor (EMERGE study) in patients with metastatic OG and CRC Significance ctDNA has potential for wide ranging applications in early and metastatic GI malignancies to directly impact patient care

Elucidating Prostate Cancer Biology Through Integrative Computational Multi-omic Analyses

in this thesis, I aimed to elucidate the microbial landscape of castration-resistant prostate cancer (CRPC), focusing on novel biomarkers of therapeutic resistance. We employed metagenomic sequencing of over 200 CRPC patients, alongside in vitro cell Line and ex vivo patient-derived xenograft (POX), models, to investigate microbial biomarkers of progression from hormone-sensitive prostate cancer {HSPC) to CRPC. Metagenomic sequencing effectively characterised the gut microbiome ofCRPC patients. While microbiome alpha diversity between HSPC and CRPC I remained similar, species-level changes, including an increased abundance of Adlercreutzia equolifaciens, were observed in CRPC. Further, L- 1 citrulline biosynthesis by the microbiota was linked to improved AR -negative prostate cancer cell viability, highlighting its potential role in CRPC i progression. Analysis ofCRPC biopsy samples provided inconclusive evidence for intra-tumour micro biota presence, with contamination confounding microbial classifications. Single-nucleus sequencing revealed significant myeloid cell heterogeneity in CRPC, with varying degrees of oxidative stress-induced DNA damage I linked to myeloid-derived reactive oxygen species {ROS). This heterogeneity extended to tumour subclones, with genomic instability and replication I stress driving therapeutic sensitivity to ATR inhibition. Notably, the overexpression of the prognostic marker POLQ was associated with replication [ stress and ATR inhibition sensitivity, highlighting a potential therapeutic target for CRPC. These findings provide insights into the complex interplay: between the microbiome, inflammation, and cellular mechanisms driving CRPC progression and resistance and merit further functional validations

Mechanisms of replication stress: the role of RAD52 in replication and a new vulnerability in mismatch repair deficient cancers

Faithful duplication of chromosomes is critical for maintaining survival and preventing genomic instability. However, both endogenous and exogenous factors can disrupt the DNA synthesis process, leading to replication stress (RS). In turn, RS leads to the accumulation of genetic modifications causing genomic instability, a hallmark of tumorigenesis. Understanding the causes and consequences of replication stress is critical, not only for furthering the knowledge of the mechanistic landscape of tumorigenesis but also for the development of better anti-cancer strategies targeting cancer vulnerabilities. The first part of the thesis explores the consequences of RAD52 knockout in non-cancerous (RPE1) and cancerous cells (HeLa and HCT116) under conditions of low-dose aphidicolin (APH) treatment and translesion synthesis inhibition (TLSi). While TLSi alone reduces global DNA synthesis in cancer cells, it has minimal impact on untransformed cells. Notably, combined TLSi and APH treatment exacerbates the DNA synthesis defect in all cell lines, highlighting a requirement for TLS in mitigating RS. Interestingly, RAD52 loss is synergistic with these effects only in HCT116 cells. Treatment with APH leads to underreplicated regions that complete DNA synthesis in G2. Further, analysis of these G2 DNA synthesis events reveals that TLS plays a critical role in maintaining replication at these regions. The data also shows RAD52 to be necessary in HCT116, but dispensable in RPE1 cells. Investigation using chemical inhibitors and siRNA-mediated depletion of RAD52 indicates that the phenotypes observed in HCT116 are likely due to compensatory mechanisms rather than direct functional loss of RAD52. Additionally, a potential synthetic lethal interaction between MLH1 loss and TLSi was identified, warranting further exploration. The biological complexities in studying the functional role of RAD52 across various cell types and conditions led to a shift in focus towards exploring novel synthetic lethality relationship between mismatch repair (MMR) deficiency and TLSi in the second part of the thesis. This lethality was confirmed across multiple untransformed cell lines (RPE1, RPE1P53KO, 1BR3, MCF10A). Notably, MMR-deficient colon cancer cell lines exhibit increased sensitivity to TLSi compared to MMR-proficient colorectal cancers. A proof-of-concept analysis of the TCGA cohort further demonstrates that this interaction could be exploited in gastrointestinal and endometrial cancers. At a molecular level, it is demonstrated that combined loss of MMR and TLS leads to ssDNA accumulation, increased p21 levels, heterochromatinization and cell cycle delays. Furthermore, new insights reveal that loss of MMR genes alone modulates cellular homeostasis, significantly impacting chromatin compaction, replication speed, and cell cycle progression

Proteogenomic discovery of RB1-defective phenocopy in cancer predicts disease outcome, response to treatment, and therapeutic targets.

Genomic defects caused by truncating mutations or deletions in the Retinoblastoma tumor suppressor gene (RB1) are frequently observed in many cancer types leading to dysregulation of the RB pathway. Here, we propose an integrative proteogenomic approach that predicts cancers with dysregulation in the RB pathway. A subset of these cancers, which we term as "RBness," lack RB1 genomic defects and yet phenocopy the transcriptional profile of RB1-defective cancers. We report RBness as a pan-cancer phenomenon, associated with patient outcome and chemotherapy response in multiple cancer types, and predictive of CDK4/6 inhibitor response in estrogen-positive breast cancer. Using RNA interference and a CRISPR-Cas9 screen in isogenic models, we find that RBness cancers also phenocopy synthetic lethal vulnerabilities of cells with RB1 genomic defects. In summary, our findings suggest that dysregulation of the RB pathway in cancers lacking RB1 genomic defects provides a molecular rationale for how these cancers could be treated

Fitness and transcriptional plasticity of human breast cancer single-cell-derived clones.

Clonal fitness and plasticity drive cancer heterogeneity. We used expressed lentiviral-based cellular barcodes combined with single-cell RNA sequencing to associate single-cell profiles with in vivo clonal growth. This generated a significant resource of growth measurements from over 20,000 single-cell-derived clones in 110 xenografts from 26 patient-derived breast cancer xenograft models. 167,375 single-cell RNA profiles were obtained from 5 models and revealed that rare propagating clones display a highly conserved model-specific differentiation program with reproducible regeneration of the entire transcriptomic landscape of the original xenograft. In 2 models of basal breast cancer, propagating clones demonstrated remarkable transcriptional plasticity at single-cell resolution. Dichotomous cell populations with different clonal growth properties, signaling pathways, and metabolic programs were characterized. By directly linking clonal growth with single-cell transcriptomes, these findings provide a profound understanding of clonal fitness and plasticity with implications for cancer biology and therapy

Development and implementation of efficient adaptive designs in early phase oncology trials for targeted agents

Recent advancements in molecular biology and genomic research have led to the development of a new class of cancer treatments known as targeted agents. Unlike cytotoxic therapies, which work by directly killing cancer cells, targeted agents focus on specific molecular markers that drive tumour growth. This distinct mechanism presents significant challenges to the traditional early-phase clinical trial design, which was originally tailored for cytotoxic treatments. This thesis focuses on three pivotal challenges posed by targeted therapies to the conventional design paradigm. Key approaches have been proposed to address these challenges. The occurrence of late-onset toxicities and rapid patient recruitment in early-phase trials highlights the challenge of incomplete toxicity information. We conducted a methodological review of existing designs that accommodate incomplete toxicity and evaluated the implementation of these designs in published trials in Chapter 2. Our findings underscore the importance of reporting sufficient information to ensure trial replicability while addressing the issue of incomplete toxicity data. To support more robust decision-making under such conditions, we propose the incorporation of the Dose Transition Pathways (DTP) look ahead strategy tool with two time-to-event toxicity designs in Chapter 3. This approach aims to balance the trade-off between accelerated dose escalation and recruitment suspension when toxicity outcomes are still pending. Non-monotonic dose-efficacy relationships and delayed responses further complicate the use of traditional designs. In Chapter 4, we introduce the DTP-TITE-CFO-ET design, which accounts for delayed toxicities, delayed efficacy, as well as non-monotonic dose-efficacy relationship. We also extend the DTP look-ahead strategy to the joint outcomes setting with incomplete information. This method allows for more informed and timely dose decisions, enhancing the flexibility and applicability of advanced early phase trial designs for targeted therapies. In Chapter 5, we propose a novel two-stage single-arm design that uses progression-free survival (PFS) as an intermediate endpoint in the interim analysis and overall survival (OS) in the final analysis. By employing a multi-state survival model to link PFS and OS, we improve the statistical power of trials while providing a framework for early stopping when appropriate. Collectively, the designs presented in this thesis offer a comprehensive approach to overcoming the limitations of traditional early-phase clinical trials when applied to targeted therapies. These advancements not only address the design challenges introduced by targeted agents but also enhance decision-making through real-time data integration and advanced statistical method

Impact of NICE Guideline NG241 'Ovarian Cancer: identifying and managing familial and genetic risk' on a regional NHS family history and clinical genetics service

BackgroundNICE Guideline NG241: identifying and managing familial and genetic risk of ovarian cancer (OC) was published by the National Institute for Health and Care Excellence (NICE) in March 2024. NG241 advises germline genetic testing of genes predisposing to OC in unaffected individuals with an OC family history at different mutation likelihood thresholds depending on age and sex, ranging from 2% to 10% likelihood of finding a germline pathogenic variant (GPV). Prior to implementation of NG241, updates to the NHS England National Genomic Test Directory would be required. Clinical genetics services have to consider equity of access to assessment and testing across all familial cancer types, best use of their limited resources and other factors such as complexity of delivery of clinical pathways.MethodsWe analysed data from 8011 patients who provided digital family histories to the South West Thames Centre for Genomics between October 2019 and June 2024.ResultsWe estimate 527/782 (68%) females and 28/77 (36%) males would meet test criteria for NICE NG241. We estimate we would reject 2919/5485 (53%) females and 135/1208 (11%) males with the same likelihood of carrying a GPV, but with a breast cancer rather than OC family history. Testing the familial OC cohort at a universal 5% threshold in OC families would detect ~11 carriers for 229 tests compared with ~8 carriers for 278 tests following NG241 criteria.ConclusionOur data highlight additional factors needing to be considered before the NICE Guideline NG241 can be implemented by regional genetics services.</jats:sec

Evolutionary and immune microenvironment dynamics during neoadjuvant treatment of esophageal adenocarcinoma.

Locally advanced esophageal adenocarcinoma remains difficult to treat and the ecological and evolutionary dynamics responsible for resistance and recurrence are incompletely understood. Here, we performed longitudinal multiomic analysis of patients with esophageal adenocarcinoma in the MEMORI trial. Multi-region multi-timepoint whole-exome and paired transcriptome sequencing was performed on 27 patients before, during and after neoadjuvant treatment. We found major transcriptomic changes during treatment with upregulation of immune, stromal and oncogenic pathways. Genetic data revealed that clonal sweeps through treatment were rare. Imaging mass cytometry and T cell receptor sequencing revealed remodeling of the tumor microenvironment during treatment. The presence of genetic immune escape, a less-cytotoxic T cell phenotype and a lack of clonal T cell expansions were linked to poor treatment response. In summary, there were widespread transcriptional and environmental changes through treatment, with limited clonal replacement, suggestive of phenotypic plasticity

Low-coverage whole genome sequencing of low-grade dysplasia strongly predicts advanced neoplasia risk in ulcerative colitis.

BACKGROUND: The risk of developing advanced neoplasia (AN; colorectal cancer and/or high-grade dysplasia) in ulcerative colitis (UC) patients with a low-grade dysplasia (LGD) lesion is variable and difficult to predict. This is a major challenge for effective clinical management. OBJECTIVE: We aimed to provide accurate AN risk stratification in UC patients with LGD. We hypothesised that the pattern and burden of somatic genomic copy number alterations (CNAs) in LGD lesions could predict future AN risk. DESIGN: We performed a retrospective multicentre validated case-control study using n=270 LGD samples from n=122 patients with UC. Patients were designated progressors (n=40) if they had a diagnosis of AN in the ~5 years following LGD diagnosis or non-progressors (n=82) if they remained AN-free during follow-up. DNA was extracted from the baseline LGD lesion, low-coverage whole genome sequencing performed and data processed to detect CNAs. Survival analysis was used to evaluate CNAs as predictors of future AN risk. RESULTS: CNA burden was significantly higher in progressors than non-progressors (p=2×10-6 in discovery cohort) and was a very significant predictor of AN risk in univariate analysis (OR=36; p=9×10-7), outperforming existing clinical risk factors such as lesion size, shape and focality. Optimal risk prediction was achieved with a multivariate model combining CNA burden with the known clinical risk factor of incomplete LGD resection. Within-LGD lesion genetic heterogeneity did not confound risk prediction. CONCLUSION: Measurement of CNAs in LGD is an accurate predictor of AN risk in inflammatory bowel disease and is likely to support clinical management